Ion-exchange is a chromatographic method frequently used for isolation of compounds with ionic or ionizable groups. Different compounds can be separated from each other on the basis of their net charge. Like in all chromatographic methods, two mutually immiscible phases are brought into contact, wherein one phase is stationary and the other is mobile. Thus, a sample that comprises one or more target compounds is introduced into the mobile phase, where it undergoes a series of interactions between the stationary and the mobile phases as it is being carried through the system by the mobile phase. During the desorption or elution step, separated compounds emerge in the order of increasing interaction with the stationary phase. The least retained component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
The stationary phase of an ion-exchanger usually comprises two components, namely a polymer matrix and functional groups coupled thereon. Such functional groups are commonly denoted ligands. The ligands are permanently bonded ionic groups that have counterions of opposite charge. These counter-ions can be exchanged for an equivalent number of other ions of the same sign in the mobile phase. Thus, in cation-exchange methods, the ligands are of negative charge, while in anion-exchange methods, the ligands are of positive charge. Proteins are normally positively charged at low pH values and negatively charged at high pH. Hence, both cation-exchange and anion-exchange techniques can be used in protein separations. In cases where negatively charged DNA is an important contaminant, e g in processing of cell homogenates, cation exchange processes are preferred in order to specifically adsorb the protein component.
Important factors in the choice of a suitable matrix for ion-exchange are, inter alia, the adsorption capacity obtained, and the selectivity and specificity of the ligands. For practical reasons, there is also a need of general matrices that can be used for example for separation of many kinds of proteins.
Coating of chromatographic base matrices with various polymers has been disclosed. Thus, U.S. Pat. No. 5,030,352 (Purdue Research Foundation) discloses a method of rendering a rigid hydrophobic polymer surface hydrophilic, which surface is useful on a chromatographic medium. More specifically, a hydrophobic surface, such as a polystyrene divinylbenzene polymer material, is exposed to a solute that adsorbs via a hydrophobic domain leaving hydrophilic domains extending from the surface. Said extending domains are subsequently cross-linked in place to produce a coating which is sufficiently hydrophilic to partially or completely mask the hydrophobic surface. The solute is defined by having short, interdispersed, hydrophilic and hydrophobic domains, and is illustrated with the two monomers epichlorohydrin and glycidol. Since the polymerisation is performed under conditions of cation polymerisation, the epichlorohydrin will act as a hydrophobic chlorine-functional comonomer and not as a cross-linker during the polymerisation. The coated polymer is described as erosion resistant, compatible with aqueous protein solutions and chemically stable at most pH values. If desired, the coating can comprise groups that can be further derivatised to produce a chromatography material, such as an ion-exchanger. However, since the coating has been adsorbed via hydrophobic/hydrophilic interactions, it will form a comparatively flat dense layer on the pore surfaces. The most satisfactory coatings were obtained with glycidol/epichlorohydrin ratios below 3, while ratios of 5-10 are stated to be less advantageous. Thus, the flat coating obtained, which is comprised of a surface of cross-linked polymer which is not covalently coupled to the support, is advantageous in the given case where the purpose of the coating is to prevent interactions between proteins and the hydrophobic pore surfaces. However, the flat dense surface will prove less advantageous for uses wherein a high diffusion rate and a high capacity are desired.
Similar to the above, U.S. Pat. No. 5,503,933 (Purdue Research Foundation) discloses hydrophilic coatings covalently bound to hydrophobic surfaces as well as methods for their production. To form the coated surfaces, a compound is provided which comprises a hydrophobic domain covalently and flexibly bonded to a hydrophilic domain, wherein the hydrophobic domain comprises an unsaturated group. Said compound is adsorbed onto the hydrophobic surface, and the unsaturated groups in its hydrophobic domains are then covalently cross-linked to the unsaturated groups on the surface by a free radical reaction. This method serves the same purpose as the above-discussed U.S. Pat. No. 5,030,352 and also gives a similarly flat, dense and also cross-linked layer. As mentioned above, this kind of layers is not the most advantageous from the diffusion point of view.
It has also been suggested to provide chromatographic matrices, wherein porous base matrices are treated with polymer in order to fill the pores with polymer. Thus, U.S. Pat. No. 5,906,747 (Biosepra Inc) discloses chromatographic media characterised by high static and dynamic sorption capacity, which are also said to exhibit improved chemical stability at alkaline and basic conditions and reduced tendencies to cause non-specific protein adsorption. This is achieved by treating a porous matrix with a passivating mixture of a main monomer, which comprises a vinyl monomer having at least one polar substituent, a passivating monomer, which comprises hydrophobic domains, e.g. a long-chain saturated hydrocarbon, an olefinic hydrocarbon group, or an aromatic group, and a cross-linker. The preferred matrices are porous mineral oxide particles. The method disclosed in U.S. Pat. No. 5,906,747 results in a composite material, wherein a polymeric gel network is confined within the pores of the matrix. The confinement of the polymeric gel network will prevent any substantial swelling of the gel, which is stated to be undesired since it “dilutes” the number of binding sites available and hence reduces its binding capacity. Still, the composite matrix disclosed allows solutes to move freely within the entire polymeric network while interacting electrostatically with more than one group present thereon. However, the use of this kind of products is limited, since many protein types do not penetrate into the hydrogel structure to the desired extent.
WO 99/64149 discloses another hydrogel product for adsorption purposes. More specifically, a support matrix, such as a protein or agarose, is coated with at least two layers of polyalkylene amine, such as polyethylene amine. Said core is subsequently removed by degradation, e.g. enzymatically or by hydrolysis. The invention is illustrated in the context of removal of undesirable metal ions from a leachate.
Another technology for pore filling of chromatographic matrices is described in U.S. Pat. No. 5,114,577 (Mitsubishi Kasei Corp.). More specifically, a composite separating agent is disclosed, which is comprised of an organic porous polymer substrate in the pores of which a hydrophilic polymer, which exhibits a giant network structure, has been deposited. The organic polymer substrate is made from a synthetic copolymer of a monounsaturated monomer, such as styrene, and a polyunsaturated monomer, such as divinylbenzene. An illustrative hydrophilic polymer is dextran, e.g. cross-linked with epichlorohydrin, which has a molecular weight Mw of about 500,000 before cross-linking. Thus, the dextran is allowed to diffuse into the porous substrate and is then cross-linked in the pore system by addition of epichlorohydrin. The resulting composite separating agent exhibits an excellent permeability of liquids and is therefore intended for use in gel permeation chromatography (GPC). The degree of cross-linking of the substrate should be 4-100% in order to provide a sufficient mechanical rigidity therefore. The resulting composite separating agent presents a macro-network structure reminiscent of the above-discussed U.S. Pat. No. 5,906,747, where the pores of the support matrix are completely filled with a hydrogel of crosslinked polysaccharide. In the GPC applications intended by U.S. Pat. No. 5,114,577, the hydrogel will prevent larger molecules from entering the macropores of the support. It is expected that if this hydrogel were to be derivatised to form an ion exchanger, it would only work for those particular proteins that partition favourably into the hydrogel.
Further, in order to provide chromatographic separation matrices with enhanced binding capacity, alternative coatings comprised of extenders that together provide a fluffier layer have been suggested. For example, WO 98/33572 (Amersham Pharmacia Biotech AB) discloses a method for adsorbing a substance from a liquid in a fluidised bed or in a stirred suspension comprised of such matrices.
An illustrative example of such a porous or fluffy layer comprised of extenders is illustrated in WO 00/75195 (Amersham Pharmacia Biotech AB), wherein a method of hydrophilisation or surface area enlargement of a porous base matrix is disclosed. More specifically, polyhydroxy polymers carrying a plurality of —(CH2CH2O)nH (polyethylene glycol) groups are attached to a porous base matrix, either via grafting of ethylene oxide or by coupling of an etoxylated polymer such as ethoxylated polyvinyl alcohol. Polyethylene glycol is in itself a highly linear molecule, while ethoxylated polyvinyl alcohol is characterised by a structure of a comb-like polymer, i.e. a linear core with short side chains of polyethylene glycol.
Another kind of extenders is disclosed in WO 95/13861, wherein in principle linear extenders comprised of poly(vinyl ether) are suggested.
The technique of using a fluffy layer as a coating to modify and enhance the binding capacities of chromatographic matrices has also been applied in commercial products. For example, Sepharose™ XL (Amersham Biosciences AB, Uppsala, Sweden) is a product that comprises an agarose matrix grafted with a layer of dextran to increase the availability of ion-exchange ligands coupled thereto. The dextran, which is derived from Leuconostoc mesenteroides, strain B512-F, is of medium molecular weight, such as about 40 kD, and is medium branched, meaning that about 5% of the glucose residues are branching points, giving a DB of 0.1.
Royappa: J Appl Polym Sci 65, 1897 (1997) reports an examination of boron trifluoride-catalyzed cationic copolymerisation of epichlorohydrin and glycidol with reference to the effect of various reaction variables, such as temperature and water content. The pattern of monomer consumption indicates the formation of a block or graft copolymer, with some branching and generation of small ring species. Thus, similar to the above-discussed U.S. Pat. No. 5,030,352, the resulting polymer will be a hydrophobic-hydrophilic polymer wherein chlorine remaining from epichlorohydrin is still present. It is also mentioned that the copolymers produced can be coated onto microscopic porous cross-linked poly(styrene-divinylbenzene) beads for use in chromatography. Such cross-linked beads are in their uncoated form extremely hydrophobic, but when coated with this polymer, the beads are rendered hydrophilic and water-wettable. The coating provides useful reactive groups on the bead surface, such as the hydroxyl group from the glycidol, which can be further derivatised to create different kinds of chromatographic media. However, in affinity chromatography and ion exchange, the hydrophobic parts of the polymers can still be expected to give rise to unspecific adsorption, which is usually undesired.
Further, Royappa et al (Journal of Applied Polymer Science, Vol. 82, 2290-2299 (2001): Amphiphilic Copolymers of Glycidol with Nonpolar Epoxide Comonomers) reports an investigation of copolymers of glycidol with various comonomers, such as epichlorohydrin, synthesised by cationic ring-opening polymerisation. The comonomer product consists of a hyperbranched polyglycidol core and has a low molecular weight. These products are useful as HIC coatings without need of any further purification. It is summarised that bot NMR and FTIR data are consistent with highly branched polyether chains replete with hydroxyl groups and side groups from the comonomers. However, from the spectral data, it does not appear that any of the side groups in the copolymer participates in the reaction in any way, which may prove a drawback for certain applications.
WO 96/31549 discloses a step-wise method of producing dendrimeric graft polymers. More specifically, the invention discloses dendrimeric graft polymers based on carriers containing hydroxyl groups on the surfaces of which polymers are covalently bound by an end-position monomer unit to the carrier. Hence, each structure will be tethered to the carrier at one point.
Finally, Cherestes and Engel (POLYMER Volume 35, Number 15, 1994:3343-3344) describe dendrimeric ion exchange materials. More specifically, ion exchange materials are disclosed wherein dendrimeric “balloons” or “strings” have been attached to a polymer backbone. The “ballons” are dendrimers elaborated in several directions from a core site, with one non-branching unit bound to the core. Thus, such dendrimers, also known as cascade molecules, are species incorporating elements of repetitive symmetry. The dendrimers disclosed contains multiple cationic sites incorporated covalently into a single structural unit. The materials are produced from styrene/divinylbenzene copolymer treated with a tertiary amine reagent.
In summary, in the field of chromatography, there is still a need of alternative base matrices, which after derivatisation with desired ligands can provide efficient ion-exchangers useful for efficient isolation of a larger range of various proteins.